Regional differences in fat accumulation must develop from imbalances in fatty acid uptake or free fatty acid (FFA) release between fat depots. Our goal is to discover the etiology of regional differences in fat distribution between different types of obesity. We found that direct storage of circulating FFA independent of lipoprotein lipase seems to best relate to body fat distribution. During this funding cycle we sought to determine which steps in lipogenesis best relates to direct FFA storage by measuring direct FFA storage in lean and obese adults with a wide range of body fat distribution, as well as visceral adipose tissue FFA storage and the effects of sex steroids on adipose tissue fatty acid metabolism. We made the surprising discovery that plasma FFA concentrations are the best, and often the only, positive predictor of adipocyte FFA storage rates. This implies that increased adipocyte lipolysis and increases uptake and storage of circulating FFA go together despite an unfavorable concentration gradient. The cellular factors related to adipocyte lipogenesis that we measured: 1) facilitated inward transport (CD36, FATP-1);2) activation of fatty acids via acyl-CoA synthetase (ACS);3) triglyceride (TG) synthesis -mediated by diacylglycerol acetyltransferase (DGAT) were poorly predictive of FFA storage, and thus almost certainly not rate limiting. Our data leads us to propose that at low plasma FFA concentrations facilitated inwards fatty acid transport is the rate limiting step for fatty acid storage whereas the rate-limiting step at high plasma FFA concentrations may be an intracellular trafficking step. Recent findings regarding the heterogeneity of different sized adipocytes within the same tissue bed also suggests there could be heterogeneity with respect to fatty acid storage between large and small adipocytes. The specific aims of this proposal are to: 1. Assess whether direct FFA storage in subcutaneous adipose tissue is greater in small vs. large adipocytes within the same depot. 2. Determine whether adipose tissue FFA storage rates plateau at physiologically maximal plasma FFA concentrations, and if so whether maximal storage is related to transport (CD36/FATP-1), activation (ACS) or TG synthesis DGAT). 3. Examine whether variations in adipose tissue lipogenic factors, induced by weight loss or pioglitazone, alter regional FFA storage under conditions of low plasma FFA concentrations. 4. Examine whether alterations in adipose tissue lipogenic factors, induced by weight loss or pioglitazone, alter regional FFA storage under conditions of physiologically maximal plasma FFA concentrations. Completing these studies should provide firm evidence as to what types of cellular/molecular processes account for the physiologic observations and lead to productive models to improve regional fat storage from the perspective of metabolic health. PUBLIC HEALTH RELEVANCE: People who gain fat mostly in their abdomen are more likely to develop diabetes and other health problems. These studies are to better understand why adipose tissue in some parts of the body stores fat better than fat in other parts of the body. We will measure how fat storage is regulated by specific fat cell proteins in humans according to their body shape, hormone status, and whether they take medications for diabetes.